Glycosyltransferase GnT-V having neovascularization action

ABSTRACT

The present invention provides a peptide or protein having a neovascularization action and containing a basic amino acid cluster region of β1,6-N-acetylglucosaminyltransferase, a neovascularization accelerator containing the above-mentioned peptide or protein, a method of screening an inhibition substance for the above-mentioned peptide or protein, and a neovascularization inhibitor containing this inhibition substance.

FIELD OF THE INVENTION

The present invention relates to a secretory type glycosyltransferase,neovascularization action of N-acetylglucosaminyltransferase V(hereinafter, abbreviated as GnT-V), a basic amino acid cluster of theGnT-V relating to the neovascularization action, a utilization of GnT-Vas neovascularization accelerator, a method of screening inhibitor forGnT-V and the basic amino acid cluster of GnT-V, a substance obtained bythis screening method, a method of screening a substance inhibitingproduction of secretory type GnT-V, a substance obtained by thisscreening method, and a utilization of this substance as aneovascularization inhibitor.

BACKGROUND ART

In growth of cancers, factors such as fibroblast growth factor-2(FGF-2), vascularendothelial growth factor (VEGF) and interleukin-8(IL-8) and the like are involved. Production of these factors andcytokines is controlled by complicated mechanisms such as increase ingene expression, modification after translation of gene products, mutualaction with extracellular matrix, and so on.

Many growth factors and receptors thereof are glycoproteins, and some ofthem are involved in neovascularization in tumor tissue. Recent studiesusing glycosyltransferase genes have revealed that change in thestructure of an oligosaccharide of a growth factor receptor causesvariation of intracellular signal transmission, leading to cancerizationof cells (Yamashita, K., et al., J. Biol. Chem. 260, 3963-3969 (1985).Pierce, M & Arango, J., J. Biol. Chem. 261, 10772-10777(1986). Zhu, T.Y., et al., J. Cancer Res. Clin. Oncol. 123, 296-299 (1997). Petretti,T., et al., Gut 46, 359-366 (2000)). It is suggested thatβ1,6-N-acetylglucosaminyltransferase V (GnT-V) catalyzing formation ofβ(1,6) branch of asparagine sugar chain is the most importantglycosyltransferase involved in metastasis of cancers (Demetriou, M., etal., J. Cell Biol. 130, 383-392 (1995). Dennis, J. W., et al., Science236, 582-585 (1987)).

Neovascularization is an essential stage in progress of cancers such asmetastasis and growth of cancers (Folkman, J., N. Eng. J. Med. 285,1182-1186 (1971). Folkman, J. Ann. Surg. 175, 409-416 (1972)). A recentstudy using transgenic mouse lacking in GnT-V directly showed that GnT-Vis essential for the growth of cancers and metastasis of cancers(Granovsky, M., et al., Nature Med. 6, 306-312 (2000)). Clinical studieshave indicated increase in GnT-V activity in malignant tumors in lungand liver. It is shown that, in human lung cancer cells, GnT-V activityand size of tumors have a positive correlation (Dennis, J. W. & Laferte,S., Cancer Res. 49, 945-950 (1989)), and it is clarified that expressionof GnT-V in human colon cancer cells is related with poor prognosis andmetastasis (Murata, K., et al., Clin. Cancer Res. 6, 1772-1777(2000)).However, detailed mechanisms of growth and metastasis of cancers viaGnT-V have not been clarified yet.

Asparagine type sugar chains (Asn type sugar chains) found inglycoproteins are classified into three types of high mannose type,composite type and mixed type depending on its constituent sugars andtype of branching. Biosynthesis of these Asn type sugar chains initiatesfirst by one time transfer of sugar chain portions from a lipidintermediate into asparagine of a polypeptide chain under translation,in the lumen side of rough endoplasmic reticula. Thereafter, glucose andsome mannoses are removed in rough endoplasmic reticula, however, someglycoproteins having an Asn type sugar chain localizing in roughendoplasmic reticula remain as they are, to leave high mannose typesugar chains. Other organelle glycoproteins, cell surface glycoproteinsor secretory glycoproteins move to a Golgi body by vesicletransportation, and mannose is removed. In this Golgi body,N-acetylglucosamine is introduced by the action ofN-acetylglucosaminyltransferase groups which are Golgi body enzymes togive a branch structure. By formation of this branch structure,conversion from a high mannose type sugar chain into a mixed type sugarchain and a composite type sugar chain initiates, and throughintroduction of fucose and introduction of galactose in a trans-Golgiregion, finally, sialic acid is introduced to complete biosynthesis ofAsn type sugar chains.

It is known that various enzymes act as a catalyst in each step of thesequential Asn type sugar chain synthesis. SixN-acetylglucosaminyltransferases are known as enzymes catalyzing areaction of introducing transfer of N-acetylglucosamine in the formationof various branch structures of Asn type sugar chains in these steps.Schachter et al. (Brockhausen, I., et al., Biochem. Cell Biol., 66, 1134(1988)) referred these six enzymes transferring N-acetylglucosamine intoa core structure of a trimannosyl structure of Man α1-3 (Man α1-6) Manβ1-4 GlcNAc β1-4 GlcNAc as GnT-I to GnT-VI. Of them, GnT-V is an enzymerelating to formation of β(1,6) branch structure (-[GlcNAc β(1,6) Manα(1,6) Man]-). It is known that the β(1,6) branch structure is presentin remarkably increased amount in cell transformation strains andtumor-forming cells (Pierce, M., et al., Biochem. Biophys. Res. Commun.,146, 679-684 (1987) and Arango, J., & Pierce, M., J., Cell. Biochem.,257, 13421-13427 (1982)). Further, it is shown that there is a relationbetween cancer metastasis ability of tumor-forming cells and emergenceof a β(1,6) branch (Hiraizumi, et al., A., Arch. Biochem. Biophys. 280,9-19 (1990)). It is reported that in human, emergence of a β(1,6) branchis accentuated in 50% of cases who received biopsy of breast carcinoma(Dennis, J. W., & Laferte, S. Cancer Res. 49, 945-950 (1989)). It isknown that in any cases, emergence of a β(1,6) branch structure isfollowed by increase in GnT-V activity. Thus, GnT-V is an enzyme whichis important not only in catalysis of formation of a β(1,6) branchstructure in sugar chain biosynthesis route but also in relation with aeasy transfer ability and malignancy of cancer cells.

SUMMARY

The present invention has been made in view of the above-mentionedconditions and an object thereof is to provide a new therapeutic targetrelating to cancer metastasis and growth which are most importantproblems in cancer therapy, and a therapeutic agent, a screening methodof finding therapeutic agents, an evaluation method and a diagnosismethod by clarifying a role played by a glycosyltransferase GnT-V oncancer metastasis and growth. Also, the present invention provides a newtherapeutic idea that inhibition of secretion or expression of GnT-Vsuppresses not only cancer metastasis but also neovascularization whichis a factor relating to cancer enlargement at metastasis site byproviding a new biochemical concept that GnT-V promotes cancermetastasis and neovascularization. Further, the present inventionprovides a new drug design target in various ischemic diseases due toblood circulation disorder caused by vascular damage and the like, ifneovascularization is regarded as a positive factor.

The present inventors have found that GnT-V which is one ofglycosyltransferases has an action of accelerating neovascularizationwhich is an initial regulation stage in cancer metastasis and subsequentcancer growth, as a new function utterly different from the originalfunction as a glycosyltransferase. Namely, secretory type GnT-V andrecombinant GnT-V which is purified promote in vitro and in vivoneovascularization at physiological concentration. Further, the presentinventors have confirmed that a basic amino acid cluster regioncontaining a significant amount of basic amino acids showing an actionof releasing a fibroblast growth factor (FGF-2) from heparan sulfateproteoglycan (HSPG) on the surface of cells and in extracellular matrixis present in amino acid sequences of GnT-V. One of the presentinvention is a peptide or protein having an amino acid sequence in abasic amino acid cluster region of GnT-V, and a neovascularizationaccelerator containing this peptide or protein.

The present inventors have found that a glycosyltransferase GnT-V and apeptide having an amino acid sequence in a basic amino acid clusterregion of this glycosyltransferase (basic peptide) accelerateneovascularization by releasing FGF-2 from HSPG on cancer cell surface,thereby promoting cancer metastasis and growth. On the basis of thesefindings, the present invention provides a method of screening acompound inhibiting neovascularization by GnT-V and the above-mentionedbasic peptide, a compound obtained by said screening method, and aneovascularization inhibitor containing said compound. Morespecifically, the present invention provides a method of screening thefollowing substances, a compound obtained by said screening method, anda neovascularization inhibitor containing said compound.

(a) A substance inhibiting a neovascularization action by GnT-V and abasic peptide

(b) A substance which inhibits a protease cutting mature GnT-V presentin a Golgi body to convert this into a secretory type GnT-V

(c) A substance inhibiting gene expression of GnT-V

(d) A substance inhibiting release of FGF-2 from heparan sulfateproteoglycan by GnT-V and a basic peptide

(e) A substance inhibiting secretion of secretory type GnT-V out of acell

Namely, the present invention relates to

(1) A peptide or protein having a neovascularization action andcontaining a basic amino acid cluster region ofβ1,6-N-acetylglucosaminyltransferase,

(2) The peptide or protein according to (1), wherein theβ1,6-N-acetylglucosaminyltransferase has the following properties:

(i) Action: N-acetylglucosamine is converted into α-6-D-mannoside usingUDP-N-acetylglucosamine as a doner substrate;

(ii) Substrate specificity: If the substrate specificity when GnGn-bi-PAis a receptor is 100%, the substrate specificity when GnGnF-bi-PA is areceptor is about 78%, the substrate specificity when GnGnGn-tri-PA is areceptor is about 125%, and the substrate specificity when GnM-PA is areceptor is about 66%;

(iii) Optimum pH: 6.2 to 6.3;

(iv) Activity: Mn²⁺ is not necessary for exertion of activity, andactivity is not inhibited even in the presence of 20 mM EDTA;

(v) Molecular weight: About 73,000 (by SDS-PAGE in the absence of areducing agent) and about 73,000 and about 60,000 (by SDS-PAGE in thepresence of a reducing agent);

(vi) Km value: Km values for a receptor GnGn-bi-PA and a donorUDP-GlcNAc are 133 μM and 3.5 mM, respectively;

(vii) having the following peptide fragments:

(a) Thr-Pro-Trp-Gly-Lys,

(b) Asn-Ile-Pro-Ser-Tyr-Val,

(c) Val-Leu-Asp-Ser-Phe-Gly-Thr-Glu-Pro-Glu-Phe-Asn-His-Ala-Asn-Tyr-Ala,

(d) Asp-Leu-Gln-Phe-Leu-Leu, and

(e) Asn-Thr-Asp-Phe-Phe-Ile-Gly,

(3) The peptide or protein according to (1), wherein theβ1,6-N-acetylglucosaminyltransferase has an amino acid sequencecontaining at least an amino acid sequence encoded by SEQ ID NO: 6, oran amino acid sequence obtained by modification of one or more aminoacids in this amino acid sequence,

(4) The peptide or protein according to (1), wherein, in the basic aminoacid cluster region, the number of basic amino acids accounts for 30% ormore of the total number of amino acids in said region,

(5) The peptide or protein according to (1), wherein the basic aminoacid cluster region contains at least an amino acid sequence as depictedin SEQ ID NO: 7, or an amino acid sequence obtained by modification ofone or more amino acids in this amino acid sequence,

(6) A neovascularization accelerator containing the peptide or proteinaccording to any of (1) to (5),

(7) The neovascularization accelerator according to (6), wherein it is awound healing agent, or an arteriosclerosis preventing and/ortherapeutic agent,

(8) A neovascularization inhibitor screening method, which comprisesusing the peptide or protein according to any of (1) to (5),

(9) A neovascularization inhibitor screening method, which comprisesusing a cell capable of secreting the peptide or protein according toany of (1) to (5) expressed in the cell out of the cell,

(10) The screening method according to (9), wherein the cell is a cellin which the peptide or protein according to any of (1) to (5) can behighly expressed,

(11) A neovascularization inhibitor screening method, which comprisesusing a protease cutting a mature typeβ1,6-N-acetylglucosaminyltransferase anchored on a Golgi body membraneto convert this into a secretory typeβ1,6-N-acetylglucosaminyltransferase,

(12) The screening method according to (11), wherein the protease isβ-secretase,

(13) The screening method according to (11), wherein the protease isγ-secretase,

(14) A compound showing a neovascularization inhibiting action in thescreening method according to any of (8) to (13),

(15) A compound showing a neovascularization inhibiting action, whereinthe compound suppresses expression of the peptide or protein accordingto any of (1) to (5),

(16) A compound showing a neovascularization inhibiting action, whereinthe compound suppresses binding of the peptide or protein according toany of (1) to (5) to heparan sulfate proteoglycan,

(17) A neovascularization inhibitor comprising the compound according toany of (14) to (16),

(18) A neovascularization inhibitor comprising a compound having aγ-secretase inhibiting action,

(19) The neovascularization inhibitor according to (18), wherein thecompound having a γ-secretase inhibiting action is a compoundrepresented by the following formula (1):

(wherein, Boc represents a butoxycarbonyl group, OMe represents amethoxy group, Val represents a valine, and Ile represents isoleucine),

(20) An antibody to the peptide or protein according to any of (1) to(5).

(21) An assay method for the peptide or protein according to any of (1)to (5), which comprises using the antibody according to (20),

(22) A detection kit for the peptide or protein according to any of (1)to (5), which comprises the antibody according to (20).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a view showing the differentiation and growth of HUVEC treatedwith culture solution of each cell, in terms of the amount ofincorporation of [³H]-thymidine as an index. CRT is a normal freshmedium used for culture of HUVEC.

FIG. 2 is a view showing a relation between the addition amount ofGnT-VΔ73 and the differentiation and growth of HUVEC.

FIG. 3A is a schematic view of an amino acid sequence of eachGnT-V-deficient variant. FIG. 3B is a view showing an HUVECdifferentiation and growth-accentuating action by each GnT-V-deficientvariant.

FIG. 4 is a view showing similarity between an amino acid sequence of abasic cluster region of GnT-V and amino acid sequences of VEGF₁₈₉,P1GF-2 and HB-EGF.

FIG. 5 is a view showing the amount of discharge of FGF-2 by variousdeficient variants of GnT-V and a synthetic peptide.

FIG. 6 is a view showing an HUVEC differentiation andgrowth-accentuating action by various deficient variants of GnT-V and asynthetic peptide.

FIG. 7 is a schematic view showing induction of cancerneovascularization by GnT-V. Secretory type GnT-V containing a basicamino acid cluster region binds, in competition with FGF-2, to HSPG onthe surface of a cell, and resultantly, release of FGF-2 occurs, tostimulate a FGF-2 receptor on the target cell.

FIG. 8 is a view showing the outline of the structure of a mature typeGnT-V. A cut part when the mature type GnT-V is cut in the lumen of aGolgi body to be converted into a secretory type GnT-V is enlarged, andits amino acid sequence is shown.

FIG. 9A shows a GnT-V activity of a cell (PS-1 ΔE9) in which variantpresenilin-1 is highly expressing and a cell (control) in which variantpresenilin-1 is not highly expressing, in culture solution. FIG. 9Bshows a ratio of a GnT-V activity in culture solution (extracellular) toa GnT-V activity in a cell, of the above-mentioned two kinds of cells.

FIG. 10A shows a value of GnT-V activity in culture solution of aPaCa-2/GnT-V cell in the case of no addition, in the case of addition ofDMSO, and in the case of addition of DFK167 dissolved in DMSO. FIG. 10Bshows a value of GnT-V activity in culture solution of a KB/GnT-V cellin the above-mentioned three cases.

DETAILED DESCRIPTION

The present invention provides a neovascularization acceleratorcontaining a peptide or protein containing a basic amino acid clusterregion of β1,6-N-acetylglucosaminyltransferase.

The above-mentioned β1,6-N-acetylglucosaminyltransferase may be a knownsubstance, and it is, however, preferable that said enzyme has thefollowing enzymological properties.

(a) Action: N-acetylglucosamine is converted into α-6-D-mannoside fromUDP-N-acetylglucosamine;

(b) Substrate specificity: If the substrate specificity when GnGn-bi-PAis a receptor is 100%, the substrate specificity when GnGnF-bi-PA is areceptor is about 78%, the substrate specificity when GnGnGn-tri-PA is areceptor is about 125%, and the substrate specificity when GnM-PA is areceptor is about 66%;

(c) Optimum pH: 6.2 to 6.3;

(d) Activity: Mn²⁺ is not necessary for exertion of activity, andactivity is not inhibited even in the presence of 20 mM EDTA;

(e) Molecular weight: About 73,000 (by SDS-PAGE in the absence of areducing agent) and about 73,000 and about 60,000 (by SDS-PAGE in thepresence of a reducing agent);

(f) Km value: Km values for a receptor GnGn-bi-PA and a donor UDP-GlcNAcare 133 μM and 3.5 mM, respectively;

(g) having the following peptide fragments:

-   (i) Thr-Pro-Trp-Gly-Lys (SEQ ID NO: 1),-   (ii) Asn-Ile-Pro-Ser-Tyr-Val (SEQ ID NO: 2),-   (iii)    Val-Leu-Asp-Ser-Phe-Gly-Thr-Glu-Pro-Glu-Phe-Asn-His-Ala-Asn-Tyr-Ala    (SEQ ID NO: 3),-   (iv) Asp-Leu-Gln-Phe-Leu-Leu (SEQ ID NO: 4), and-   (v) Asn-Thr-Asp-Phe-Phe-Ile-Gly (SEQ ID NO: 5).

In the present invention, it is preferable to use GnT-V as theabove-mentioned β1,6-N-acetylglucosaminyltransferase. GnT-V is an enzymeinvolved in the formation of β(1,6) branch structure (-[GlcNAc-β(1,6)Man-α (1,6) Man]-). Particularly, it is preferable that theabove-mentioned β1,6-N-acetylglucosaminyltransferase has an amino acidsequence containing at least an amino acid sequence encoded by SEQ IDNO: 6, or an amino acid sequence obtained by modification of one or moreamino acids in this amino acid sequence. It is more preferable that theabove-mentioned enzyme has an amino acid sequence described inNishikawa, et al., Biochem. Biophys. Res. Commun. 198, 318-327 (1994).

The above-mentioned enzyme can be easily obtained by known methods. Forexample, human origin GnT-V can be obtained by isolating GnT-V from arat kidney and purifying this by the method described in Shoreibah, M.,et al., J. Biol. Chem. 267, 2920-2927 (1992). It can be isolated andpurified from concentrated liquid of a protein-free culture supernatantof human lung cancer (small cell carcinoma) origin QG cells by themethod described in Japanese Patent Application Laid-Open (JP-A) No.6-197756. The human lung cancer (small cell carcinoma) origin QG cell isnamed Human lung carcinoma SBM331, and internationally deposited withNational Institute of Advanced Industrial Science and Technology (AIST),International Patent Organism Depositary (IPOD) under an acceptancenumber FERM BP-3967 on Aug. 18, 1992 based on Budapest Treaty.

The peptide or protein contained in the neovascularization acceleratorof the present invention contains a basic amino acid cluster region ofthe above-mentioned β1,6-N-acetylglucosaminyltransferase, preferablyGnT-V. The above-mentioned basic amino acid cluster region indicates aportion containing significant amount of basic amino acids in which thetotal number of amino acids is from about 5 to 50, preferably from about8 to 40, more preferably from about 10 to 30. In the above-mentionedbasic amino acid cluster region, it is preferable that the number ofbasic amino acids accounts for about 30% or more, preferably from about35 to 95%, more preferably from about 40 to 90% of the total number ofamino acids in the above-mentioned region.

More preferably, the above-mentioned basic amino acid cluster regioncontains at least an amino acid sequence as depicted in SEQ ID NO: 7.The above-mentioned basic amino acid cluster region may also contain atleast an amino acid sequence obtained by modification of one or moreamino acids in the amino acid sequence as depicted in SEQ ID NO: 7.Specifically, various modification type basic amino acid cluster regionsare listed such as (a) a peptide obtained by adding one or more aminoacids to the amino acid sequence as depicted in SEQ ID NO: 7, andmaintaining a neovascularization action; (b) a peptide obtained byremoving one or more amino acids from the above-mentioned amino acidsequence, and maintaining a neovascularization action; (c) a peptideobtained by substitution of one or more amino acids in theabove-mentioned amino acid sequence by other amino acids, andmaintaining a neovascularization action; further (d) a peptide having acombination of the above-mentioned amino acid addition modification,amino acid removal modification and amino acid substitutionmodification, and maintaining a neovascularization action; and the like.The number of amino acids subjected to the above-mentioned modificationsuch as amino acid addition, removal and substitution is notparticularly restricted, and determined depending on the object of themodification, and specifically, it is about 30% or less, preferablyabout 20% or less, more preferably about 10% or less of the number ofamino acids in the basic amino acid cluster region. It is preferablethat the above-mentioned amino acid modifications such as addition,removal and substitution are conducted on moieties other than basicamino acids.

The neovascularization accelerator according to the present inventionmay be the above-mentioned peptide or protein itself which is an activeingredient, however, usually, it is produced by mixing this activeingredient with a pharmaceutically acceptable carrier by a method knownper se. [methods commonly used in the field of formulation technologies,for example, methods described in the Japanese Pharmacopoeia (forexample, 13th edition) and the like]. The dosage form of theneovascularization accelerator according to the present inventionincludes, for example, oral preparations such as tablets (includingcoated tablets such as sugar-coated tablet or enteric tablet andmulti-layer tablet), capsules (including soft capsules, microcapsules),powders, granules, syrups and the like, and parenteral preparations suchas injections (for example, subcutaneous injection, intravenousinjection, intramuscular injection, intraperitoneal injection and thelike), external preparations (for example, intranasal preparation,percutaneous preparations such as ointment), suppositories (for example,rectal suppository, vaginal suppository and the like), pellets, drops,sustained-release preparations (for example, sustained-releasemicrocapsule and the like) and the like. The neovascularizationaccelerator according to the present invention is preferably aparenteral preparation.

As the pharmaceutically acceptable carrier, various organic or inorganiccarrier substances commonly used as raw materials in the formulation areused, and listed are excipients, lubricants, binders and disintegratingagents in solid preparations; and solvents, solubilizing agents,suspending agents, isotonizing agents, buffers, soothing agents and thelike in liquid preparations. If necessary, additives in the formulationsuch as preservatives, antioxidants, coloring agents, sweetening agentsand the like can also be used.

Preferable examples of the excipient include lactose, sucrose,D-mannitol, starch, crystalline cellulose, light silicic acid anhydrideand the like. Preferable examples of the lubricant include magnesiumstearate, calcium stearate, talc, colloidal silica and the like.Preferable examples of the binder include crystalline cellulose,sucrose, D-mannitol, dextrin, hydroxypropylcellulose,hydroxypropylmethylcellulose, polyvinylpyrrolidone and the like.Preferable examples of the disintegrating agent include starch,carboxymethylcellulose, carboxymethylcellulose calcium, crosscarmellosesodium, carboxymethyl starch sodium and the like.

Preferable examples of the solvent include water for injection, alcohol,propylene glycol, macrogol, sesame oil, corn oil and the like.Preferable examples of the solubilizing agents include polyethyleneglycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol,trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodiumcitrate and the like. Preferable examples of the suspending agentinclude surfactants such as stearyltriethanolamine, sodium laurylsulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride,benzethonium chloride, glycerin monostearate and the like; hydrophilicpolymers such as polyvinyl alcohol, polyvinylpyrrolidone,carboxymethylcellulose sodium, methylcellulose, hydroxymethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose and the like. Preferableexamples of the isotonizing agent include sodium chloride, glycerin,D-mannitol and the like. Preferable examples of the buffer includebuffer solutions of phosphates, acetates, carbonates and citrates andthe like. Preferable examples of the soothing agent include benzylalcohol and the like. Preferable examples of the preservative includep-hydroxybenzoate ester, chlorobutanol, benzyl alcohol, phenethylalcohol, dehydroacetic acid, sorbic acid and the like. Preferableexamples of the antioxidant include sulfite, ascorbic acid and the like.

The neovascularization accelerator according to the present inventioncan be used for mammals (for example, human, mouse, rat, rabbit, dog,cat, bovine, horse, swine, monkey and the like).

The application of the neovascularization accelerator according to thepresent invention is not particularly restricted, and it is preferablyused as a wound healing agent. The dose of the agent in this case is notdetermined indiscriminately since it varies depending on the type ofdisease conditions to be treated, the age and body weight of patient,symptoms, the seriousness of disease and the like, but it is about 0.01to 100 mg/kg, preferably about 0.1 to 50 mg/kg. Particularly, it ispreferable that the neovascularization accelerator according to thepresent invention is locally applied, in the form of liquid, ointment,cream, gel, cataplasm and the like, to a wound region and absorbedpercutaneously to heal the wound. In the case of the external liquidpreparation, the above-mentioned peptide or protein can be applied, forexample, at a concentration of about 0.001 to 1000 mg/ml, furtherpreferably of about 0.01 to 500 mg/ml. In the case of the externalpreparation other than liquid preparations, it is preferable that theabove-mentioned peptide or protein is contained at a concentration ofabout 0.01 to 10 wt %.

The neovascularization accelerator according to the present inventioncan be used for treatment or prevention of aneurysm; arteriosclerosissuch as coronary arteriosclerosis, cerebral arteriosclerosis orperipheral arteriosclerosis; peripheral artery obstruction, acutemyocardial infarction (AMI), deep-vein thrombosis, pulmonary embolism,dissecting aneurysm, transient ischemic attack (TIA), apoplexy, andother obstruction-related disorders; unstable angina pectoris,disseminated intravascular coagulation (DIC), sepsis, surgical orinfectious shock, postoperative and postpartum trauma, cardiopulmonarybypass surgical operation, incompatible blood transfusion, prematureseparation of the placenta, thrombotic thrombocytopenia purpura (TTP),acute or chronic renal diseases due to excess agglomeration such assnake venom and immune diseases, inflammation, hemolytic-uremicsyndrome, symmetric peripheral necrosis, and bedsore. Further, theneovascularization accelerator according to the present invention can beused for enhancing the action of a thrombolytic agent and preventingre-obstruction, preventing re-obstruction after PTCA, preventingthrombocytopenia due to dialysis, preventing thrombosis caused byartificial blood vessels and organs.

When the neovascularization accelerator according to the presentinvention is used in the above-mentioned applications, the dose thereofis not determined indiscriminately since it varies depending on theapplication of the neovascularization accelerator, the type of diseaseconditions to be treated, the age and body weight of patient, symptoms,the seriousness of disease and the like, but it is about 0.01 to 100mg/kg, preferably about 0.1 to 50 mg/kg, per day. Particularly, whenadministered intravenously, the dose thereof is about 0.01 to 5 mg/kg,preferably about 0.04 to 1.5 mg/kg, per day. It is desirable that thisdose is administered 1 to 3 times per day.

In the neovascularization accelerator of the present invention, therecan be used a concomitant drug not giving an adverse effect on theneovascularization action of the peptide or protein according to thepresent invention. The concomitant drug is not particularly restrictedand when the neovascularization accelerator of the present invention isused as a treating and/or preventing agent for arteriosclerosis and thelike, examples thereof include hypotensive agents, hypolipidemic agents,diuretics, thrombolytics and the like.

The timing of administration of the neovascularization acceleratoraccording to the present invention and concomitant drug is notparticularly restricted and these may be administered simultaneously, oradministered at a time interval, to the subject to be administered. Thedose of the concomitant drug may be advantageously determined accordingto clinically used dose, and can be appropriately selected depending onthe target subject, age and body weight of the target subject, symptoms,time of administration, dosage form, administration route, combinationand the like. The administration form of the concomitant drug is notparticularly restricted, and it may be advantageous that theneovascularization accelerator according to the present invention andconcomitant drug are combined at the time of administration.

The present invention provides an antibody to a peptide or proteincontaining a basic amino acid cluster region ofβ1,6-N-acetylglucosaminyltransferase. The above-mentioned antibody to apeptide or protein as an antigen may be any of a monoclonal antibody andpolyclonal antibody. These antibodies can be produced according to knownmethods described, for example, in “Basic Experiment Method of Proteinand Enzyme, 2nd revision (T. Horio ed., published by NANKO DO, 1994)” or“Method in Enzymology vol. 182 published by ACADEMIC PRESS, INC. 1990”and the like.

The present invention provides an assay method for the above-mentionedpeptide or protein having a neovascularization action using theseantibodies, and a detection kit for the above-mentioned peptide orprotein having a neovascularization action using this assay method. Suchan assay method and detection kit can be utilized in variousapplications. For example, neovascularization is an essential process incancer metastasis, and therefore, a possibility of cancer metastasis canbe found by measuring the presence or absence or the amount of theabove-mentioned peptide or protein having a neovascularization action inthe blood or cancer tissue of a patient with cancer using the assaymethod and detection kit according to the present invention.

In the assay method and detection kit according to the presentinvention, an antibody molecule itself may be used, and alternatively,F(ab′)₂, Fab′ or Fab fractions of the antibody molecule may be used. Inthe assay method and detection kit according to the present invention,an antibody of GnT-V or a fraction thereof is preferably used.

For the above-mentioned assay method and production of detection kit,known methods can be used. For example, as the method for quantifyingthe above-mentioned peptide or protein having a neovascularizationaction using the above-mentioned antibody, there are mentioned ameasurement method in which the amount of an antibody corresponding tothe antigen amount (for example, protein amount) in a test solution, theamount of an antigen or an antibody-antigen complex are detected bychemical or physical means, and this is calculated from the standardcurve produced by using a standard solution containing an antigen inknown amount, and other methods. More specifically, nephrometry,competition method, immunometric method or sandwich method, for example,are suitably used, and it is particularly preferable to use a sandwichmethod described later from the standpoint of sensitivity andspecificity.

Specific embodiments of the assay method according to the presentinvention will be described below, however, the scope of the inventionis not limited to these embodiments. Namely, there is exemplified, asthe above-mentioned assay method, (i) a method of quantifying theabove-mentioned peptide or protein having a neovascularization action ina test solution, wherein an antibody to the above-mentioned peptide orprotein having a neovascularization action, a test solution, and theabove-mentioned peptide or protein having a neovascularization actionwhich has been labeled (hereinafter, referred to as simply “labeledpeptide” in this column) are allowed to react competitively, and theproportion of the labeled peptide bound to the antibody is measured.Then, there is also mentioned (ii) a method of quantifying a peptide orprotein having a neovascularization action in a test solution in which;the antibody to the above-mentioned peptide or protein having aneovascularization action is held on a carrier to provide insolubility;the antibody to the peptide or protein having a neovascularizationaction recognizing a region other than the above-mentioned insolubilizedantibody is labeled; next, the test solution, the antibody insolubilizedon the carrier, and the labeled antibody are reacted simultaneously orsequentially; then, the activity of the labeling agent arrested via theantigen (peptide or protein having a neovascularization action) on thecarrier and/or the activity of the labeling agent not arrested on thecarrier is measured. Further, as the method of assaying the peptide orprotein having a neovascularization action of the present invention,detection by tissue stain and the like can also be conducted in additionto quantification of the peptide or protein having a neovascularizationaction using a monoclonal antibody to the peptide or protein.

As the labeling agent used in such an assay method according to thepresent invention, for example, radioactive isotopes, enzymes,fluorescent substances, light-emitting substances and the like arelisted. As the above-mentioned radioactive isotope, for example, ¹²⁵I,¹³¹I, ³H or ¹⁴C and the like are used. As the above-mentioned enzyme,those which are stable and having large specific activity arepreferable, and examples thereof include β-galactosidase, β-glucosidase,alkaline phosphatase, peroxidase, malic acid dehydrogenase and the like.As the above-mentioned fluorescent substance, for example,fluorescamine, fluorescein isothiocyanate and the like are used. As theabove-mentioned light-emitting substance, for example, luminol, luminolderivatives, luciferin, lucigenin and the like are used. Furthermore, abiotin-avidin system can also be used for binding of an antibody orantigen with a labeling agent.

The present invention provides a neovascularization inhibitor. Theneovascularization inhibitor according to the present invention ischaracterized in that it comprises one or more compounds selected fromthe group consisting of (a) a compound showing a neovascularizationinhibiting action in the above-mentioned screening method using apeptide or protein having a neovascularization action, (b) a compoundinhibiting the activity of a protease cutting a mature typeβ1,6-N-acetylglucosaminyltransferase anchored on a Golgi body membraneto convert this into a secretory typeβ1,6-N-acetylglucosaminyltransferase, (c) a compound suppressingexpression of the above-mentioned peptide or protein having aneovascularization action, and (d) a compound suppressing binding of theabove-mentioned peptide or protein having a neovascularization action toheparan sulfate proteoglycan. The neovascularization inhibitor accordingto the present invention may also contain (e) a compound inhibitingsecretion of the above-mentioned peptide or protein having aneovascularization action out of a cell. The above-mentioned compound(e) can also be used in combination with one or more of theabove-mentioned compounds (a) to (d). The above-mentioned compounds (a)to (e) will be described in detail below.

The above-mentioned compound (a) showing a neovascularization inhibitingaction in a screening method using a peptide or protein having aneovascularization action can be obtained by a screening method asdescribed below. Namely, as this screening method, there is mentioned amethod in which neovascularization is observed in the case of thepresence of the above-mentioned peptide or protein having aneovascularization action and a test substance in a system of observingneovascularization described in detail in examples, and it is comparedwith neovascularization in the case of the absence of a test substance.When neovascularization in the case of the presence of a test substanceis smaller as compared with neovascularization in the case of theabsence of a test substance in such a screening method, such a testsubstance is recognized as a substance showing a neovascularizationinhibiting action. In this screening method, it is preferable to useGnT-V as the peptide or protein having a neovascularization action. Morespecifically, it may be advantageous that the total length ofnewly-produced blood vessels measurable from a micrograph in the case ofthe presence of a test substance is about 90% or less, preferably about80% or less, more preferably about 70% or less, based on that in thecase of the absence of a test substance. In a method of evaluatingneovascularization described in detail in examples, it may beadvantageous that the value in the case of the presence of a testsubstance is about 90% or less, preferably about 80% or less, morepreferably about 70% or less, based on the value in the case of theabsence of a test substance.

Here, the test substance used in the above-mentioned screening method isnot particularly restricted, and may be a protein, or a compound of lowmolecular weight, or a compound of high molecular weight. It may also bea purified substance, or a mixture containing several co-existentcompounds. Further, it may also be that of natural origin such asculture solution of a microorganism or a chemically synthesizedsubstance. Moreover, the test substance may be a novel compound or aknown compound. These are applied also in the following screeningmethod.

The compound (b) inhibiting the activity of a protease cutting a maturetype GnT-V anchored on a Golgi body membrane to release this from aGolgi body membrane and to convert this into a secretory type GnT-V canbe easily obtained by a screening method using the above-mentionedprotease. The screening method using the above-mentioned protease is notparticularly restricted, and when the secretory type GnT-V generated byallowing the above-mentioned protease to act on the mature type GnT-Vanchored on a Golgi body membrane in the presence of a test substance issmaller as compared with that obtained in the absence of a testsubstance, such a test substance is recognized as a compound inhibitingthe activity of a protease. The above-mentioned test substanceinhibiting the activity of a protease may also be screened in a testtube.

As the above-mentioned protease, for example, β-secretase and the likeare listed. The amino acid sequence of the β-secretase is described inVassar, R., et al., Science 286, 735-741 (1999) and the like, and easilyavailable from information of GenBank accession number AF190725 and thelike. As the above-mentioned protease, γ-secretase is also mentioned.γ-secretase cuts, at a transmembrane site, a carboxyl terminal peptidefragment, bound to a Golgi body membrane, of a 12 KD amyloid proteinproduced by cutting of an amino terminal of an amyloid-β-precursor byβ-secretase (Tsai, J. Y., et al., Curr. Med. Chem. 9, 1087-1106 (2002)).Though the entity of γ-secretase is not clarified yet, it is believed toform a composite with presenilin bound to a Golgi body membrane and tocut amyloid protein (Wolfe, M. S., Curr. Top. Med. Chem. 2, 371-383(2002)). The cell expressing γ-secretase can be obtained by knownmethods described in the above-mentioned literatures. For example, therecan be used cells (e.g., PS-1 ΔE9) having a γ-secretase activityenhanced by highly expression of presenilin, and the like.

As the compound (b), for example, compounds having a γ-secretaseinhibiting action are listed, and such compounds may have any structureproviding they have an action of suppressing or inhibiting a γ-secretaseactivity. As the compound having a γ-secretase inhibiting action, thereare listed compounds represented by the following formula (1):

(wherein Boc represents a butoxycarbonyl group, OMe represents a methoxygroup, Val represents valine, and Ile represents isoleucine).Even derivatives of such compounds can be used as the neovascularizationinhibitor in the present invention so long as they have a γ-secretaseinhibiting action. As the derivative of the above-mentioned compoundrepresented by the formula (1), there are listed, for example, (a)compounds in which a methyl group is substituted by a C₂-C₆ lower alkylgroup, (b) compounds in which a butoxycarbonyl group which is aprotective group at the amino terminal of valine is converted to aprotective group of another amino group, (c) compounds in which amethoxy group which is a protective group at the carboxyl terminal ofisoleucine is converted to a protective group of another carboxyl group,(d) compounds obtained by reducing the above-mentioned compoundrepresented by the formula (1) to convert one or two carbonyl groups toa —CHOH— group, (e) compounds in which one or two valines are convertedto another amino acid, preferably an aliphatic amino acid such asglycine, alanine, leucine or isoleucine and the like, (f) compounds inwhich one or two isoleucines are converted to another amino acid,preferably an aliphatic amino acid such as glycine, alanine, leucine orvaline and the like, (g) compounds having a combination of two or moreconversions in (a) to (f), and the like.

The compound (c) suppressing expression of the above-mentioned peptideor protein having a neovascularization action can be obtained by knownmethods. For example, a method using a promoter for expression of theabove-mentioned peptide or protein having a neovascularization actionand a reporter gene (T. Yokota, K. Arai, Biomanual series 4, YODO sha(1993)) is mentioned. Namely, the compound (c) can be obtained by ascreening method using a transformant containing an introduced promoterfor expression of the above-mentioned peptide or protein having aneovascularization action, preferably a promoter of a GnT-V gene, and anintroduced reporter gene. More specifically, the above-mentionedpromoter is connected to a translation region of the reporter gene toproduce an expression vector, this expression vector is introduced intoa host cell to produce a transformant, this transformant is cultured fora certain time, then, any amount of a test substance is added, and theamount of the reporter expressed by the cell after a certain time ismeasured as an enzyme activity, or as the amount of the expressedprotein. More specifically, when the expression amount of the reportergene in the presence of a test substance is smaller as compared with theexpression amount of the reporter gene in the absence of a testsubstance, such a test substance can be recognized as a substancesuppressing expression of the above-mentioned peptide or protein havinga neovascularization action.

In the above-mentioned method, it is preferable to use a promoter regionupstream of a GnT-V gene as the promoter for expression of theabove-mentioned peptide or protein having a neovascularization action.Such a promoter can be obtained by cloning 5′-upstream region of a GnT-Vgene from a genome of an HuCC-T1 cell (Saito, H., et al., Eur. J.Biochem. 233, 18-26 (1995)). The HuCC-T1 cell can be obtained fromJapanese Cancer Resources Bank.

In the above-mentioned method, any genes encoding peptides or proteinsmay be used as the reporter gene so long as the activity or productionamount of the expressed product (also including the production amount ofmRNA) can be measured by persons skilled in the art. For example,chloramphenicol acetyltransferase (CAT), β-glactosidase (β-Gal),luciferase and the like can be utilized by measuring enzymatic activity.Secretory type growth hormone and the like can be utilized by measuringits production amount by an immune antibody reaction method and thelike.

The above-mentioned expression vector can be obtained by inserting atranslation region of the above-mentioned promoter and reporter genesinto a replicable vector. The replicable vector is not particularlyrestricted, and pUC18 or pGEM-3Z and the like are listed as thosereplicable in E. coli. The above-mentioned expression vector isintroduced into a host cell to produce a transformant. The host cell isnot particularly restricted and can be selected appropriately dependingon the type of the expression vector. Such transformation can beconducted by usual methods. As the transformant used in the presentinvention, those in which an expression vector is transiently introducedinto a host are also used, in addition to those in which an expressionvector is stably introduced into a host chromosome. Selection of thetransformants in which an expression vector is stably introduced into ahost chromosome can be conducted by transforming a host cell with avector in which a selection marker gene is introduced into a vector tobe introduced, or a vector containing a selection marker simultaneouslywith a vector to be introduced, and culturing the transformed cell in amedium in which only that having a selection marker can survive.

More preferably, a compound suppressing expression of theabove-mentioned peptide or protein having a neovascularization actioncan be obtained by the following method. Namely, (a) DNA containing atleast one of the following base sequence: 5′-GGGAGTGAGGATGATGTAGGGAAG-3′(SEQ ID NO: 8) and 5′-ATGGGGCAGAGGAACTTACGTTAT-3′ (SEQ ID NO: 9); (b) anEts-1 protein or fragment thereof; and (c) a test substance areincubated together, and binding of the above-mentioned DNA (a) with anEts-1 protein or fragment thereof is measured.

Transcription of a GnT-V gene is promoted by binding of an Ets-1 proteinto a specific site shown in the above-mentioned sequence in a promoterregion upstream of a GnT-V gene. The peptide or protein contains atleast a basic amino acid cluster region of GnT-V. Therefore, a testsubstance inhibiting binding of DNA with an Ets-1 protein or fragmentthereof in the above-mentioned sequence can suppress expression of theabove-mentioned peptide or protein having a neovascularization action.

As the method of measuring binding of DNA with an Ets-1 protein orfragment thereof in the above-mentioned sequence, known methods may beused. As preferable embodiments of such a measuring method, a gel shiftassay and supershift assay are listed and these methods will beillustrated in detail below.

The gel shift assay is conducted, for example, as described below.5′-extended terminal of DNA in the above-mentioned sequence is labeledusing [γ-³²P] DATP (available, for example, from Amersham). The resulted³²P labeled DNA (10,000 cpm) and a cut Ets-1 protein or nuclear extractwhich is in vitro transcribed/translated of MOLT4 cell are mixedtogether with a buffer containing 65 mM KCl, 25 mM Tris-HCl (pH 7.9), 6mM MgCl₂, 0.25 mM EDTA and 10% glycerol so that the total volume is 20ml. Subsequently, 2 μg of poly(dI-dC) (available, for example, fromSigma) is added to the reaction mixture. Then, the reaction mixture iscultured for 1 hour at room temperature. The resulted culture solutionis added on 6% non-denaturing polyacrylamide gel(acrylamide:bisacrylamide=29:1), 0.5×TBE (1×TBE=89 mM Tris, 89 mM boricacid, 2 mM EDTA), then, electrophoresis is conducted at 40° C. and 150 Vfor 1 hour. After electrophoresis, the gel is dried by a gel drier, andthen, exposed to an X-ray film (available, for example, from Kodak).

In the gel shift assay, the mobility manifested by the composite of DNAof the above-mentioned sequence with an Ets-1 protein or fragmentthereof in electrophoresis using non-denaturing polyacrylamide geldecreases as compared with that manifested by DNA of the above-mentionedsequence not bound to an Ets-1 protein or fragment thereof. When a testsubstance is added in given amount to the above-mentioned reactionmixture, if a band of a composite of DNA of the above-mentioned sequencewith an Ets-1 protein or fragment thereof is not observed or its bandquantity decreases in the result of electrophoresis obtained by theabove-mentioned procedure, the test substance can be judged to be asubstance inhibiting binding of DNA of the above-mentioned sequence withan Ets-1 protein or fragment thereof.

The supershift assay is conducted in the same manner as for the gelshift assay, except that anti-Ets-1 IgG (available, for example, fromCambridge Research Biochemicals) not cross-reacting with a protein inother Ets family is added to the reaction mixture. In the supershiftassay, the mobility manifested by the composite of DNA of theabove-mentioned sequence with an Ets-1 protein or fragment thereof inelectrophoresis using non-denaturing polyacrylamide gel decreases ascompared with that manifested by DNA of the above-mentioned sequence notbound to an Ets-1 protein or fragment thereof, to a greater extent thanin the gel shift assay. When a test substance is added in given amountto the above-mentioned reaction mixture, if a band of a composite of DNAof the above-mentioned sequence with an Ets-1 protein or fragmentthereof is not observed or its band quantity decreases in the result ofelectrophoresis obtained by the above-mentioned procedure, the testsubstance can be judged to be a substance inhibiting binding of DNA ofthe above-mentioned sequence with an Ets-1 protein or fragment thereof.

The compound (d) suppressing binding of the above-mentioned peptide orprotein having a neovascularization action to heparan sulfateproteoglycan may also be a compound which decreases affinity of theabove-mentioned peptide or protein having a neovascularization actionwith heparan sulfate proteoglycan, as well as a compound preventingbinding of the above-mentioned peptide or protein having aneovascularization action to heparan sulfate proteoglycan. Theabove-mentioned peptide or protein having a neovascularization actionbinds to heparan sulfate proteoglycan on the surface of a cell or on anextracellular matrix in competition with FGF-2 (fibroblast growthfactor-2). Further, since the above-mentioned peptide or protein havinga neovascularization action has higher affinity, than that of FGF-2, toheparan sulfate proteoglycan, FGF-2 bound to heparan sulfateproteoglycan is dissociated from heparan sulfate proteoglycan. Thusgenerated free FGF-2 stimulates an enthothelium to causeneovascularization. Therefore, if there is a compound which suppressesbinding of the above-mentioned peptide or protein having aneovascularization action to heparan sulfate proteoglycan or whichdecreases affinity with heparan sulfate proteoglycan, FGF-2 can binddominantly to heparan sulfate proteoglycan and the process ofneovascularization as described above does not progress.

As the compound suppressing binding of the above-mentioned peptide orprotein having a neovascularization action to heparan sulfateproteoglycan, for example, compounds blocking a basic amino acid clusterregion of this peptide or protein, and the like are listed. Specificexamples of such compounds include peptides and proteins containing anacidic amino acid cluster region containing a significant amount ofacidic amino acids. Preferable as the above-mentioned acidic amino acidcluster region are portions containing significant amount of acidicamino acids having a total number of amino acids of about 5 to 50,preferably about 8 to 40, more preferably about 10 to 30. In theabove-mentioned acidic cluster region, it is preferable that the numberof acidic amino acids accounts for about 30% or more, preferably about35 to 95%, more preferably about 40 to 90% of the total number of aminoacids in the above-mentioned region. Preferable as the compound (d) arecompounds suppressing binding of GnT-V to heparan sulfate proteoglycan.

The compound (e) inhibiting secretion of the above-mentioned peptide orprotein having a neovascularization action out of a cell can be easilyobtained by a screening method using a cell capable of secreting apeptide or protein having a neovascularization action expressed in acell out of the cell. Specifically, there is mentioned a-screeningmethod in which the above-mentioned cell is cultured in the presence ofa test substance, and the amount of a peptide or protein having aneovascularization action secreted in the culture solution is measured.In such a screening method, if the amount of a peptide or protein havinga neovascularization action decreases, the test substance can be aneovascularization inhibitor.

“Cell capable of secreting a peptide or protein having aneovascularization action expressed in a cell out of the cell” may be acell which secretes a peptide or protein having a neovascularizationaction such as, for example, human colon cancer cell strain WiDr and thelike, preferably a secretory type GnT-V. Particularly, preferable is atransformant containing an introduced gene encoding part or all of apeptide or protein having a neovascularization action, preferably GnT-V,the cell being capable of highly expressing a peptide or protein havinga neovascularization action, preferably a secretory type GnT-V, ascompared with a wild type cell. As this cell, there are listed a cell(PaCa-2/GnT-V cell) in which GnT-V is highly expressed by introductionof a GnT-V gene into a pancreas cancer cell MIA PaCa-2, a cell (KB/GnT-Vcell) in which GnT-V is highly expressed by introduction of a GnT-V geneinto an oral cavity cancer cell KB, and the like.

The amount of the peptide or protein having a neovascularization actionsecreted into culture solution can be measured directly using, forexample, gel electrophoresis and the like. Further, the amount of thepeptide or protein having a neovascularization action secreted intoculture solution can also be measured indirectly by measuring theactivity of the peptide or protein in culture solution, for example,β1,6-N-acetylglucosaminyltransferase activity.

The neovascularization inhibitor according to the present invention maybe at least one compound itself among the above-mentioned compounds (a)to (e) which is an active ingredient, however, usually, it is producedby mixing the active ingredient with a pharmaceutically acceptablecarrier by a method known per se. [methods commonly used in the field offormulation technologies, for example, methods described in the JapanesePharmacopoeia (for example, 13^(th) edition), and the like]. Here, asthe pharmaceutically acceptable carrier, the same compounds as for theabove-mentioned neovascularization accelerator are listed. As the dosageform of the neovascularization inhibitor according to the presentinvention, the same dosage forms as those of the above-mentionedneovascularization accelerator are exemplified, and particularly, theneovascularization inhibitor according to the present invention ispreferably a parenteral preparation.

The neovascularization inhibitor according to the present invention canbe used for mammals (for example, human, mouse, rat, rabbit, dog, cat,bovine, horse, swine, monkey and the like). The dose thereof is notdetermined indiscriminately since it varies depending on the type ofactive ingredients of neovascularization inhibitors, the type of diseaseconditions to be treated, the age and body weight of patients, symptoms,the seriousness of diseases, and the like.

The application of the neovascularization inhibitor according to thepresent invention is not particularly restricted and can be used aspreventing and treating agents for various diseases includingneovascularization, for example, tumors (for example, malignantmelanoma, malignant lymphoma, digestive organ (e.g., stomach, intestineand the like) cancer, lung cancer, pancreas cancer, esophageal cancer,breast cancer, liver cancer, ovarian cancer, uterine cancer, prostatecancer, kidney cancer, bladder cancer, brain cancer, Kaposi's sarcoma,angioma, osteosarcoma, myosarcoma, angiofibroma and the like),inflammatory diseases (for example, rheumatic arthritis, psoriasis andthe like), diabetic retinopathy, atherosclerosis (including abnormalangiopoiesis by formation of abnormal capillary network inatherosclerosis nest outer membrane) and the like. Theneovascularization inhibitor of the present invention can be used alsoas an agent for treating eye hyperemia.

In the neovascularization inhibitor of the present invention, there canbe used concomitant drugs not exerting an adverse effect on theneovascularization inhibition action of the above-mentioned compounds(a) to (e). As such concomitant drugs, there are listed, for example,antitumoragents, cachexy improving agents, antidiabetic agents otherthan insulin resistant improving agents, diabetic complication treatingagents, antiobestic agents, hypotensive agents, hypolipidemic agents,diuretics and the like, and two or more of them may be combined. In useof the neovascularization inhibitor of the present invention, surgicaltherapy (operation) or radiation therapy may be conducted.

The timing of administration of the neovascularization inhibitoraccording to the present invention and concomitant drug, the dose of theconcomitant drug, and the administration form of the concomitant drugsare the same as those in the case of the above-mentionedneovascularization accelerator.

EXAMPLES

The present invention will be illustrated in detail by the followingexamples, however, the scope of the invention is not limited to theseexamples.

Example 1 Acceleration of Neovascularization in a Nude Mouse byMetastasis of GnT-V Transformant

Since expression of GnT-V shows a high correlation with metastasis andpoor prognosis of colon cancer, transformants ofβ1,4-N-acetylglucosaminyltransferase III (GnT-III) andα1,6-fucosyltransferase (FucT) as a control were produced together witha stable transformant of GnT-V using a human colon cancer cell WiDr, andthese were injected subcutaneously to a nude mouse to examine aninfluence exerted on cancer metastasis. The human colon cancer cellstrain WiDr was cultured on an RPMI-1640 medium (manufactured by GIBCOBRL.) containing 10% fetal bovine serum (FBS) and antibiotics(penicillin and streptomycin). Transformation was conducted using a CELLFECTIN (registered trademark) reagent (manufactured by GIBCO BRL.), andtransformation was conducted according to a method described in a manualof CELL FECTIN (registered trademark). Regarding transplantation of theabove-mentioned transformed cancer cell to a nude mouse, 5×10⁵ cellstransformed with the above-mentioned glycosyltransferases were injectedon the back of the nude mouse, and formation of cancer andneovascularization were visually observed one month after. Though theWiDr cell originally contains slight expression of the above-mentionedglycosyltransferase, acceleration of cancer metastasis was observed andremarkable neovascularization was observed in tumor tissue in the nudemouse transplanted with the GnT-V transformant as compared with the nudemouse transplanted with WiDr cells transformed with otherglycosyltransferase genes. This result suggests that a cancer cellexcessively expressing GnT-V secretes a certain factor acceleratingneovascularization.

Example 2 Induction of Neovascularization by GnT-V Transformant

Acceleration of neovascularization by a GnT-V transformant was confirmedby a CAM (chorioallantoic membrane) assay using an embryo of a chickenfertilized egg. The CAM assay was conducted by a method of Yen et al.(Yen, L., et al., Oncogene 19, 3460-3469 (2000)) and a method ofBernardini (Bernardini, G., et al., Blood 96, 4039-4045 (2000)), bothbeing slightly modified. CAM 8 days after fertilization of white Leghornwas used, and 1×10⁵ cells thereof were inoculated on a collagen spongeand maintained for 4 hours. A 5 mm silicon ring was placed on CAM on thecollagen sponge and maintained for 48 hours. It was found that invadingof blood cells into the collagen sponge occurred only in the case of theGnT-V transformant among WiDr cells transformed with theglycosyltransferase gene described in Example 1. Also in cells obtainedby transient transformation of a GnT-V gene into WiDr cells andnoncancerous cells such as COS-1 cell and CHO cell, acceleration ofneovascularization was observed in the CAM assay like with theabove-mentioned GnT-V stable transformant. These results stronglysuggest that a common mechanism exists for acceleration ofneovascularization by expression of GnT-V.

Example 3 Induction of Neovascularization by Culture Solution of GnT-VTransformant

For in vitro evaluation of induction of neovascularization by a GnT-Vtransformant, the amount of synthesis of DNA of human umbilical veinepithelial cells (HUVEC) after stimulation with culture solution of aGnT-V transformant was measured by a method of Soker et al.(Soker, S.,et al., J. Biol. Chem. 272, 31582-31588 (1997)). HUVEC was inoculated ona 96-well plate coated with type I collagen at a rate of 2×10³ cells perwell, and 24 hours later, the medium was substituted with an MCDB131medium (not containing FBS and FGF-2) containing 0.1% fetal bovine serumalbumin and a starved condition was maintained for 24 hours. The mediumwas substituted with culture solution of the WiDr cell transformed withthe glycosyltransferase gene described in Example 1, and HUVEC wasstimulated for 24 hours. HUVEC was maintained for 8 hours with[³H]-thymidine (1 μCi/ml), and incorporation of [³H]-thymidine intoHUVEC was analyzed by MicroBeta-Counter (manufactured by Wallac) tomeasure the amount of synthesis of DNA. The result was shown as the meanvalue of assay results of 6 wells, and the standard deviation wasmeasured. All experiments were repeated at least three times, and thesame results were obtained. As apparent from FIG. 1, DNA synthesis ofHUVEC stimulated with the culture solution of WiDr cells transformedwith a GnT-V gene increased, however, the same effect was not observedin culture solution of WiDr cells transformed with otherglycosyltransferase genes. These results indicate that the WiDr celltransformed with a GnT-V gene secretes a neovascularization factorderived from excess expression of GnT-V into culture solution.

Example 4 Influence of Recombinant GnT-V on Differentiation and Growthof HUVEC

Purification of a neovascularization factor present in culture solutionof WiDr cells transformed with a GnT-V gene was conducted using variouscolumn chromatographies. The neovascularization activity of eachfraction was evaluated by differentiation and growth of HUVEC describedin Example 3. In heparin affinity chromatography, a fraction of highgrowth activity of HUVEC was eluted with 0.3 M NaCl. Since known growthfactors such as FGF-1, FGF-2, VEGF, placenta-induced growth factor(PIGF) and hepatocyte growth factor (HGF) and the like are eluted with0.8 to 1.5 M NaCl (Hauser, S. & Weich H. A., Growth Factor 9, 259-268(1993). Gohda, E., et al., J. Clin. Invest. 81, 414-419 (1998). Marez,A., et al., Biochimie 69, 125-129 (1987). Risau, W., et al., The EMBO J.7, 959-962 (1988). Rothenthal, R. A., et al., Growth Factor 4, 53-59(1990)), the above-mentioned nature is utterly different from thenatures of these known growth factors. The WiDr cell itself does notproduce such a neovascularization factor. A fraction eluted with 0.3 MNaCl in heparin affinity chromatography was subjected to Western blotanalysis using an anti-GnT-V antibody to find that the reaction of theanti-GnT-V antibody and the differentiation and growth activity of HUVECare consistent each other, and the main protein having a differentiationand growth activity of HUVEC present in the fraction is GnT-V itself.

Though it is known that GnT-V is secreted from cancer cells (Chen, L.,et al., Glycoconjugate J. 12, 813-823 (1995)), like otherglycosyltransferases (Gu, J., et al., J. Biochm. 113, 614-619 (1993).MaCaffery, G. & Jamison, J. C., Comp. Biochem. Physiol. B. 104, 91-94(1993). Ugarte, M. A. & Rodriguez, P., J. Biochem. 23, 719-726 (1991)),the physiological significance of secretion of theseglycosyltransferases is not known. For certifying a hypothesis thatsecretory type GnT-V itself induces differentiation and growth of HUVEC,a recombinant GnT-V, called GnT-VΔ73, maintaining a glycosyltransferaseactivity but lacking in transmembrane portion was produced. The GnT-VΔ73which is a soluble recombinant GnT-V was prepared by a Baculovirussystem according to a method disclosed in a literature of Sasai et al(Sasai, K., et al., Glycobiology (in press)). As shown in FIG. 2, byaddition of GnT-VΔ73, recombinant GnT-V, differentiation and growth ofHUVEC increased in addition amount-dependent manner. The concentrationof the used GnT-VΔ73 was in the physiological range, and theconcentration of GnT-V present in culture solution of GnT-Vtransformants was 140 ng/ml based on the specific activity of GnT-VΔ73.A mouse melanoma cell of B16-F10 had a high endogenous GnT-V activity,the culture solution of B16-F10 cells contained 70 ng/ml of GnT-V, andalso the B16-F10 cell showed the same neovascularization activity in theCAM assay. Addition of recombinant Fuc-T did not show HUVEC growthaccelerating activity at all. These results show that secretory typeGnT-V in the physiological concentration range has an HUVEC growthaccelerating activity.

Example 5 Analysis of Domain of GnT-V Involved in Differentiation

For clarifying which domain of GnT-V is involved in HUVEC growthaccelerating activity, a GnT-V lacking variant shown in FIG. 3A wasproduced. The method of production of a GnT-VΔ188 plasmid is disclosedin a literature of Sasai et al (Sasai, K., et al., Glycobiology (inpress)). A transfer plasmid having a GnT-VΔ233 gene was produced bybinding a DNA fragment of 1521 base pairs encoding polyhistidine tag atthe C-terminal and an amino acid sequence of from Glu234 to Leu741 ofhuman GnT-V, obtained by cutting a GnT-VΔ188 plasmid with EcoRI andEagI, to an EcoRI-EagI site of a transfer vector pAcGP67-A (manufacturedby PharMingen). A transfer plasmid having a GnT-VΔ436 gene was producedby binding a DNA fragment of 912 base pairs encoding polyhistidine tagat the C terminal and an amino acid sequence of from Ile437 to Leu741,of human GnT-V, obtained by cutting a GnT-VΔ188 plasmid with EcoRV andEagI, to an EcoRV-EagI site of a transfer vector pAcGP67-A. Forproduction of a recombinant Baculovirus, an insect cell Sf21 wastransformed with the transfer plasmid obtained above according to amethod known in a literature (Ikeda, Y., et al., J. Biochem. 128,609-619 (2000)). The recombinant glycosyltransferase derived from thetransformed Sf21 cell was purified by Ni²⁺-chelating affinitychromatography according to a method disclosed in a literature of Sasakiet al (Sasai, K., et al., Glycobiology (in press)).

As shown in FIG. 3B, GnT-VΔ73, GnT-VΔ188 and GnT-VΔ233 variants had anHUVEC growth and differentiation accentuation action, however, GnT-VΔ436did not have an HUVEC growth and differentiation accentuation action.Though GnT-VΔ73 and GnT-VΔ188 had a glycosyltransferase activity,GnT-VΔ233 and GnT-VΔ436 had no glycosyltransferase activity. Theseresults suggest that the HUVEC growth accelerating activity is presentin a region corresponding to an amino acid sequence of from 234 to 436of GnT-V and this region does not contain a region involved in aglycosyltransferase activity.

Example 6 Identification of Basic Amino Acid Cluster Region of GnT-VInducing Neovascularization

An amino acid sequence of from 254 to 269 of human GnT-V is a sequenceof Lys-Ser-Leu-Ala-Glu-Lys-Gln-Asn-Leu-Glu-Lys-Arg-Lys-Arg-Lys-Lys (SEQID NO: 7) in which basic amino acids form a cluster, and a sequencefairly resembling this sequence is observed in an amino acid sequence offrom 142 to 157 of VEGF₁₈₉ (Hauser, S. & Weich H. A., Growth Factor 9,259-268 (1993)) (see, FIG. 4). This amino acid cluster region is alsokept in PIGF-2 and heparin binding type epidermis growth factor-likegrowth factor (HB-FGF) (see, FIG. 4), and acts as a heparin-bindingmotif (Hauser, S. & Weich, H. A., Growth Factor 9, 259-268 (1993)).Barillari et al. have reported that a basic peptide having a sequence ofGly-Arg-Gly-Lys-Arg-Arg (SEQ ID NO: 10) derived from PIGF-2 releaseFGF-2 from heparan sulfate proteoglycan (HSPG) on cell surface and/orextracellular matrix, to induce growth of epidermal cells (Barillari,G., et al., American J. Patho. 152, 1161-1166 (1998)).

A basic peptide (KRKRKK peptide) composed of Lys-Arg-Lys-Arg-Lys-Lys(SEQ ID NO: 11) which is an amino acid sequence of from 264 to 269 and anon-basic control peptide (FSGGPL peptide) composed ofPhe-Ser-Gly-Gly-Pro-Leu (SEQ ID NO: 12) which is an amino acid sequenceof from 291 to 296, of GnT-V, were synthesized, and an influence ofthese peptides on growth of HUVEC was examined. The peptide wassynthesized by a peptide synthesizer A432 (manufactured by AppliedBiosystems), and purified by reverse phase HPLC, then, its molecularweight and degree of purification were confirmed by MALDI TOF-MS(Voyager-DE (registered trademark) RP; manufactured by PerSeptiveBiosystems). The concentration of FGF-2 was measured by a known method(Barillari, G., et al., American J. Patho. 152, 1161-1166 (1998)). Thatis, HUVEC was inoculated on a 12-well plate coated with collagen at arate of 5×10⁴ cells per well, and washed twice with PBS, then, themedium was substituted with MCDB 131/0.1% BSA (0.5 ml/well), and theplate was maintained at 4° C. for 2 hours on a plate rotation table inthe presence or absence of GnT-VΔ73, GnT-VΔ436, KRKRKK peptide or FSGGPLpeptide, together with heparin. After centrifugation at 4° C. and 3000rpm for 5 minutes, the supernatant was collected, the concentration ofFGF-2 in the supernatant was measured in an FGF-2 ELISA system(manufactured by R&D Systems) according to a manual of this system.

As described above, various deficient variants of GnT-V and syntheticpeptides were added at 4° C. to culture solution of HUVEC, and theamount of FGF-2 released from HSPG on HUVEC was measured. As a result,as shown in FIG. 5, GnT-VΔ73 and KRKRKK peptide released FGF-2, however,GnT-VΔ436 and FSGGPL peptide did not affect release of FGF-2. LikeGnT-VΔ73, also GnT-VΔ188 and GnT-VΔ233 released FGF-2. Similarly,heparin (Biard, A., et al., Proc. Natl. Acad. Sci. USA 85, 2324-2328(1988)) which is known to release the HSPG binding molecule bycompetition with a heparin binding site of the HSPG binding moleculeinduced release of FGF-2. Phosphorylation of an FGF receptor on HUVEC bystimulation of released FGF-2 was also confirmed. As shown in FIG. 6,the KRKRKK peptide accelerated growth of HUVEC at the same degree asGnT-VΔ73, however, this effect was completely suppressed by ananti-FGF-2 neutralization antibody added simultaneously. These resultssuggest that a basic amino acid cluster region of GnT-V is sufficientfor an HUVEC growth accelerating activity, and a GnT-V protein releasesFGF-2 from HSPG on an endothelium by the action of a basic portion ofthis protein, to accelerate neovascularization.

Example 7 In vivo Neovascularization by GnT-V Protein

Induction of neovascularization by GnT-V was confirmed also by other invitro neovascularization assays such as a capillary-like tube formationassay (Ashoton, A. W., et al., J. Biol. Chem. 274, 35562-35570 (1999))and a migration assay (Zeng, H., et al., J. Biol. Chem. 276, 3271-3279(2001)) using HUVEC. For confirming ex-vivo neovascularization activityof GnT-V, a CAM assay using a GnT-VΔ73 protein was conducted. TheGnT-VΔ73 induced neovascularization of chicken microvessels like FGF-2,and the KRKRKK peptide induced neovascularization likewise, however,induction of neovascularization by GnT-VΔ73 and KRKRKK peptide wasinhibited by treatment with an anti-FGF-2 neutralization antibody. Incontrast, GnT-VΔ436 and FSGGPL peptide did not have a neovascularizationactivity. These results indicate that secretory type GnT-V and theKRKRKK peptide derived from GnT-V induce neovascularization via theaction of FGF-2, however, the basic region of GnT-V causes release ofFGF-2 from HSPG on an endothelium, in view of the results of the HUVECdifferentiation and growth assay.

Example 8 Analysis of Cut Site of Mature Type GnT-V Protein

As shown in FIG. 8, a mature type GnT-V has at the amino terminal side atransmembrane portion composed of a hydrophobic amino acid, andtherefore, it is believed that the amino terminal side is anchored on amembrane of a Golgi body which is an intracellular organelle, and acatalyst portion involved in a glycosyltransferase activity is presentin the lumen of a Golgi body. Since it is guessed that a mature typeGnT-V is cut in the lumen of a Golgi body to be converted into asecretory type GnT-V, and secreted out of the cell through a secretionroute, an amino acid sequence at the amino terminal of the secretorytype GnT-V secreted out of the cell was determined, and the cut part wasanalyzed.

A GnT-V gene was introduced into a pancreas cancer cell MIA PaCa-2 andGnT-V was highly expressed and this cell (PaCa-2/GnT-V) was cultured toobtain 1500 ml of serum-free culture solution. Cell transformation andculturing of transformants were conducted according to the-methodsdescribed in Example 1. This culture solution was precipitated withsaturated ammonium sulfate, and the recovered ammonium sulfateprecipitate was dissolved in 10 ml of PBS (phosphate buffer saline), anddesalted in a PD-10 column (Code Number: 17-0851-01, Amersham PharmaciaBiotech), and simultaneously, the buffer solution was substituted with50 mM Tris-HCl (pH 7.5). Subsequently, purification thereof wasconducted by affinity chromatography using, as a ligand, a mousemonoclonal antibody 24D11 of GnT-V produced according to the knownmethods described in “Basic Experiment Method of Protein and Enzyme, 2ndrevision (T. Horio ed., published by NANKO DO, 1994)” and “Method inEnzymology vol. 182 published by ACADEMIC PRESS, INC. 1990”.

A column containing Protein A Sepharose 4B as a carrier was equilibratedwith 50 mM Tris-HCl (pH 7.5) to allow a secretory type GnT-V to beadsorbed on the column, then, the secretory type GnT-V was eluted with50 mM Tris-HCl (pH 7.5) containing 0.05% of TFA (trifluoroacetic acid).The eluate was fractionated by SDS-polyacrylamide gel electrophoresis tofind a main band at a portion corresponding to a molecular weight ofsecretory type GnT-V of about 100 kD.

The amino acid sequence at the amino terminal of secretory type GnT-Vextracted from the gel was determined by a method known in theliterature. As a result, as shown in FIG. 8, the sequence at the aminoterminal of secretory type GnT-V was determined to beHis-Phe-Thr-Ile-Gln- (SEQ ID NO: 13), however, this amino acid sequencewas consistent with an amino acid sequence of from 31 to 35 in the aminoacid sequence of GnT-V encoded by SEQ ID NO: 6, therefore, it was foundthat secretory type GnT-V is produced by cutting between a 30th aminoacid leucine and a 31st amino acid histidine in mature type GnT-V.

Example 9 Identification of Protease Involved in Production of SecretoryType GnT-V and Screening of Substance Inhibiting Production of SecretoryType GnT-V

Since the cut portion in the secretory type GnT-V analyzed in Example 8is present at the boundary between a transmembrane site and a Golgi bodylumen site of GnT-V shown in FIG. 8, a γ-secretase bound to a Golgi bodymembrane was hypothesized as a protease involved in cutting.

A nerve cell SK-N-SH having a high γ-secretase activity and manifestingstrong expression of endogenous GnT-V, in which variant presenilin-1 washighly expressed, was used, and this cell was cultured in the methoddescribed in Example 1, and the influence of γ-secretase on productionof secretory type GnT-V was examined. The amount of secretion of GnT-Vin the culture solution was quantified by concentrating 10-fold theculture solution and measuring an enzymatic activity using HPLCaccording to the method described in JP-A No. 6-197756.

As shown in FIG. 9A, it was confirmed that the GnT-V activity in culturesolution of cells (PS-1 ΔE9) in which variant presenilin-1 had beenhighly expressed was about 4-fold of that of cells (control) in whichvariant presenilin-1 had not been highly expressed, and γ-secretase cutsa mature type GnT-V to produce a secretory type GnT-V. Further, as shownin FIG. 9B, it was confirmed that, in the case of the cells (PS-1 ΔE9)in which variant presenilin-1 had been highly expressed, the ratio ofthe GnT-V activity in culture solution (extracellular) to that in cellwas about 3.5, which was about 3-fold higher than a ratio in the case ofthe cells in which variant presenilin-1 had not been highly expressed ofabout 1.2, and production and secretion of secretory type- GnT-V wereaccelerated by high expression of a γ-secretase activity.

An influence of DFK167 (J. Med. Chem., 41, 6-9 (1998) known as aninhibitor for γ-secretase on production of secretory type GnT-V wasexamined. DFK167 was a compound represented by the above-mentionedformula (1) and available from ICN (Ohio, U.S.). A GnT-V gene wasintroduced into a pancreas cell MIA PaCa-2 and oral cavity cancer cellKB, and GnT-V was highly expressed to obtain cells which were culturedaccording to the method shown in Example 1, and then the activity ofGnT-V in the culture solution was measured. The GnT-V activity in theculture solution of PaCa-2/GnT-V cells to which DMSO (dimethylsulfoxide) had been added slightly decreased. As shown in FIGS. 10A and10B, the GnT-V activity could not be detected in the culture solution ofPaCa-2/GnT-V cell and KB/GnT-V cell to which DFK167 dissolved in DMSOhad been added at a concentration of 100 μM. This indicates that cuttingof GnT-V and secretion of secretory type GnT-V were inhibited completelyby DFK167 which is a γ-secretase inhibitor. Therefore, it indicates thatthe γ-secretase inhibitor such as DFK 167 is a neovascularizationinhibitor as one of the present inventions, and that the screeningmethod using a cell in which GnT-V has been highly expressed shown inthe above-mentioned examples also can be conducted as the method ofscreening a neovascularization inhibitor as one of the presentinventions.

INDUSTRIAL APPLICABILITY

The present invention provides a peptide or protein having aneovascularization action, and a neovascularization acceleratorcontaining this. This neovascularization accelerator is effective forwound healing or, for prevention and/or treatment of diseases related toarteriosclerosis, thrombosis, aneurysm, vascular obstruction.

Further, the present invention can suppress a neovascularization actionby suppressing conversion of mature type GnT-V penetrating a membraneinto secretory type GnT-V. A neovascularization action can be suppressedalso by suppressing expression of GnT-V and suppressing binding ofsecretory type GnT-V to heparan sulfate proteoglycan. Such substancessuppressing a neovascularization action are effective for preventionand/or treatment of diseases caused by neovascularization, typicallyincluding cancer metastasis and the like.

Furthermore, by use of an antibody to the above-mentioned peptide orprotein containing a basic amino acid cluster region of GnT-V, thepresence or absence or the amount of the above-mentioned peptide orprotein in a test substance can be measured, and for example, apossibility of cancer metastasis can be found.

1. An isolated polypeptide fragment of anN-acetylglucosaminyltransferase V (GnT-V), comprising a basic amino acidcluster region, wherein the basic amino acid cluster region comprisesthe amino acid sequence of SEQ ID NO: 7 and up to 50 contiguous aminoacids encoded by the sequence shown in SEQ ID NO: 6, or a variantthereof, wherein the polypeptide fragment or variant thereof possessesneovascularization activity, wherein the number of amino acids modifiedby addition, removal, or substitution in the variant is up to 10% of thenumber of amino acids in the basic amino acid cluster region, andwherein the addition, removal, or substitution is conducted on aminoacids other than basic amino acids.
 2. The polypeptide of claim 1,wherein one amino acid other than a basic amino acid of the variant isadded, removed, or substituted from the amino acid sequence encoded bythe sequence shown in SEQ ID NO:
 6. 3. The polypeptide of claim 1,wherein the number of basic amino acids accounts for greater than 30% ofthe total number of amino acids in said fragment.
 4. The polypeptide ofclaim 1, consisting of the amino acid sequence shown in SEQ ID NO:
 7. 5.A pharmaceutical composition comprising an amount of an isolatedpolypeptide consisting of SEQ ID NO: 7 sufficient to accelerateneovascularization.
 6. A pharmaceutical composition comprising an amountof the polypeptide fragment of claim 1 sufficient to accelerateneovascularization, and a pharmaceutically acceptable carrier.
 7. Apharmaceutical composition comprising an amount of the polypeptidefragment of claim 1 sufficient to promote wound healing and apharmaceutically acceptable carrier.
 8. A method for acceleratingneovascularization comprising administering an amount of the polypeptidefragment of claim 1 sufficient to accelerate neovascularization and apharmaceutically acceptable carrier therefor to a mammal.
 9. Apharmaceutical composition comprising an amount of the polypeptidefragment of claim 1 sufficient to treat arteriosclerosis and apharmaceutically acceptable carrier therefor.